TrkB receptor interacts with mGlu2 receptor and mediates antipsychotic-like effects of mGlu2 receptor activation in the mouse

Metabotropic glutamate receptor 2 (mGlu2) attracts particular attention as a possible target for a new class of antipsychotics. However, the signaling pathways transducing the effects of mGlu2 in the brain remain poorly characterized. Here, we addressed this issue by identifying native mGlu2 interactome in mouse prefrontal cortex. Nanobody-based affinity purification and mass spectrometry identified 149 candidate mGlu2 partners, including the neurotrophin receptor TrkB. The later interaction was confirmed both in cultured cells and prefrontal cortex. mGlu2 activation triggers phosphorylation of TrkB on Tyr816 in primary cortical neurons and prefrontal cortex. Reciprocally, TrkB stimulation enhances mGlu2-operated Gi/o protein activation. Furthermore, TrkB inhibition prevents the rescue of behavioral deficits by glutamatergic antipsychotics in phencyclidine-treated mice. Collectively, these results reveal a cross-talk between TrkB and mGlu2, which is key to the behavioral response to glutamatergic antipsychotics.


INTRODUCTION
Schizophrenia is a multifactorial and strongly debilitating mental disorder that affects around 1% of the world population.It is characterized by a broad range of symptoms classified as positive symptoms (e.g., hallucinations and disorder of thought), negative symptoms (e.g., social isolation and anhedonia), and cognitive deficits (e.g., poor "executive functioning" and impaired working memory) (1).Currently available antipsychotics that mainly target dopamine 2 (D 2 ) and 5-HydroxyTryptamine/serotonin 2A (5-HT 2A ) receptors show unequivocal efficiency against positive symptoms.However, they poorly control negative and cognitive symptoms and induce severe side effects, such as long-term dyskinesia and metabolic perturbations (2).During the 90s, a different class of antipsychotics acting as agonists or positive allosteric modulators of group II metabotropic glutamate receptors (mGlu 2 and mGlu 3 ) has been designed (3,4).Subsequent preclinical studies indicated that mGlu 2 activation mostly accounts for their antipsychotic activity (5,6), although implication of mGlu 3 has also recently been reported (7).These glutamatergic antipsychotics displayed promising effects against positive, negative, and cognitive symptoms in preclinical studies (8), but leading compounds failed in phase 2 and phase 3 trials.Subsequent meta-analysis of clinical data revealed an interference with broadly prescribed atypical antipsychotics (9) that was further confirmed in preclinical studies, indicating that chronic pretreatment with atypical antipsychotics greatly reduces the antipsychotic activity of mGlu 2 agonists (10).Accordingly, mGlu 2 is still considered as a target for first line treatment of psychoses such as schizophrenia, and new molecules are under clinical evaluation.mGlu 2 is canonically coupled to G i/o proteins that inhibit adenylyl cyclase (11) and calcium channels (12).However, the multiple signaling pathways transducing the effects of native mGlu 2 and underlying its antipsychotic properties remain poorly characterized.Previous findings indicated that G protein-coupled receptor (GPCR)operated signal transduction depends on their association with large protein networks.Thus, we characterized the interactome of native mGlu 2 from mouse prefrontal cortex (PFC).This region expressing high amount of mGlu 2 is involved in core symptoms of schizophrenia (13).We used a proteomic strategy combining mGlu 2 affinity purification with a DN1 version of anti-mGlu 2 nanobody (14) and systematic identification of copurified proteins by high-resolution mass spectrometry (MS) [affinity purification-MS (AP-MS)].Among the 149 identified candidates, we focused on brain-derived neurotrophic factor (BDNF)/NT-3 growth factor receptor TrkB [also designated as neurotrophic tyrosine kinase receptor type 2 (NTRK2)].Previous findings indicated a decreased expression of TrkB and its natural agonist BDNF in patients with schizophrenia and their involvement in the pathophysiology of schizophrenia (15).We explored the reciprocal influence of mGlu 2 and TrkB in their signal transduction properties and the role of TrkB in the behavioral response to glutamatergic antipsychotics in phencyclidine (PCP)-treated mice, a rodent preclinical model of schizophrenia.

Analysis of mGlu 2 interactome in mouse PFC identified 149 receptor-interacting proteins
We used a nanobody specific for mGlu 2 to immunoprecipitate endogenous receptor from mouse PFC, a brain structure involved in the effects of glutamatergic antipsychotics (13).This mGlu 2 nanobody is a biparatopic nanobody composed of two previously described mGlu 2 -specific nanobodies that displays a subnanomolar affinity (0.30 ± 0.04 nM) for mGlu 2 whatever its conformational state stabilized by a saturating concentration of either agonist or antagonist (14).The specificity of this nanobody for native mouse mGlu 2 was assessed by immunohistochemistry experiments indicating a robust immunolabelling in the brain from wild-type mice, whereas no immunolabelling was detected in mGlu 2 knockout (KO) (mGlu 2 −/− ) mice (fig.S1A).This nanobody provided high immunoprecipitation yield, as shown by the absence of remaining receptors in brain extracts after immunoprecipitation with the mGlu 2 nanobody (Fig. 1A).
Among proteins showing the highest enrichment in the mGlu 2 interactome, we focused on BDNF/NT-3 growth factor receptor TrkB (also called NTRK2, highlighted in green), given the welldocumented influence of the BDNF/TrkB axis on the pathophysiology of schizophrenia (Fig. 1B and table S1) (15).

TrkB specifically and dynamically associates with mGlu 2
Co-immunoprecipitation followed by Western blotting showed that TrkB associates with mGlu 2 in PFC from wild-type mice, but not in mGlu 2 −/− mice (fig.S1B), confirming the specificity of our nanobodybased interactomic screen.To further explore the specificity of mGlu 2 interaction with TrkB, human TrkB was coexpressed with either human Flag-tagged mGlu 2 or the closely related mGlu 3 in human embryonic kidney (HEK) 293 cells.Immunoprecipitation of mGlu receptors with a Flag antibody followed by Western blotting confirmed interaction of TrkB with mGlu 2 , while no association of TrkB with mGlu 3 was observed (Fig. 2A).Consistent with these results and further suggesting a specific association of TrkB with mGlu 2 , PLA with anti-TrkB and anti-Flag antibodies showed a PLA signal only in cells coexpressing TrkB and Flag-mGlu 2 but not in cells coexpressing TrkB and Flag-mGlu 3 (Fig. 2B).No signal was detected in cells transfected with empty vectors (Mock) or expressing either Flag-mGlu 2 or Flag-mGlu 3 or TrkB alone (Fig. 2B).Likewise, in situ PLA performed with the anti-mGlu 2 nanobody and the anti-TrkB antibody showed a close proximity of the endogenous receptors in various brain regions from the mouse known to coexpress both receptors, such as the olfactory bulb (OB), PFC, dentate gyrus (DG), and cerebellum (CB) (Fig. 2C) (20,21).PLA signals were sparse and of lower intensity in the brain from mGlu 2 −/− mice (Fig. 2C).We next assessed interaction between TrkB and mGlu 2 or mGlu 3 using bioluminescence resonance energy transfer (BRET).Under conditions of constant TrkB-Rluc expression, the BRET signal increased hyperbolically as a function of mGlu 2 -Venus expression (Fig. 2D), indicative of an interaction between both receptors.In contrast and further supporting the notion that TrkB does not associate with mGlu 3 , the BRET saturation curve in cells coexpressing TrkB-Rluc and mGlu 3 -Venus was comparable to those observed in cells coexpressing mGlu 5 -Rluc and mGlu 2 -Venus, two receptors that do not heteromerize (16), and in cells coexpressing TrkB-Rluc + CAAX-Venus as control membrane protein (Fig. 2D).The BRET saturation curve in cells coexpressing TrkB-Rluc and mGlu 2 C3-Venus, an mGlu 2 in which the C-terminal domain was replaced by C-terminal domain of mGlu 3 , was also comparable to the one observed in TrkB-Rluc + CAAX-Venus-expressing cells (Fig. 2D), indicating that the C-terminal domain of mGlu 2 is necessary for its interaction with TrkB.Note that a linear shape of the BRET curve was only observed in control cells coexpressing TrkB-Rluc and cytoplasmic Venus.

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Fig. 1.AP-MS analysis of mGlu 2 interactome in mouse PFC.(A) Representative Western blots of immunoprecipitates obtained with a nontargeting nanobody (ctrl) or an anti-mGlu 2 nanobody (mGlu 2 ) from mice PFc protein extracts.inputs represent 5% of the material used for immunoprecipitations. mGlu 2 was detected at three molecular weights that likely correspond to monomeric (100 kda), dimeric (200 kda), and higher-molecular weight complexes (more than 200 kda), respectively.(B) volcano plot illustrating the difference in protein abundance in mGlu 2 immunoprecipitates versus ctrl immunoprecipitates for each identified protein in three biological replicates.Proteins known to physically or functionally interact with mGlu 2 (GRM2, pink) are highlighted in blue.trkB (ntRK2) is highlighted in green.(C) StRinG analysis of previously reported protein-protein interactions between proteins significantly enriched in mGlu 2 immunoprecipitates (P < 0.05).connecting lines represent previously reported protein-protein interactions in public databases.Size and color of nodes correspond to the statistical significance (−log 10 of P value) of protein enrichment in mGlu 2 immunoprecipitates.M w , weight-average molecular weight; iP, immunoprecipitation; Ft, flow through of immunoprecipitation.
Administration of LY379268 (10 mg/kg, 30 min, i.p.) likewise increased p-Y 816 -TrkB immunolabeling in PFC of wild-type mice, but not mGlu 2 −/− mice (Fig. 4B).This increase in TrkB phosphorylation reached a level comparable to that measured in mice injected with 7,8-DHF (5 mg/kg, 1 hour, i.p.) (Fig. 4B).Reminiscent to our observations in cultured neurons, the LY379268 effect was abolished by pretreating mice with LY341495 (3 mg/kg, 30 min, i.p.) or with ANA-12 (2.5 mg/kg, 2 hours, i.p.).Administration of ANA-12 also prevented the increase in p-Y 816 -TrkB immunolabeling elicited by 7,8-DHF injection.Collectively, these results indicate that mGlu 2 stimulation transactivates TrkB in neurons from PFC.These findings were confirmed by Western blotting experiments showing that administration of LY379268 (0.01 to 10 mg/kg, 30 min, i.p.,) to mice   We next examined the impact of TrkB stimulation on the recruitment of G i protein to mGlu 2 by measuring the BRET signals between C-terminally Venus-tagged mGlu 2 or mGlu 3 and a chimeric Rluc8-tagged miniGα si protein (Fig. 5D) (23).As expected, treating cells with LY379268 induced a recruitment of the miniG protein directly to the mGlu receptor in cells expressing mGlu 2 (fig.S7D) or mGlu 3 alone (fig.S7E) and cells coexpressing mGlu 2 or mGlu 3 and TrkB receptor (Fig. 5, E and F).G protein recruitment elicited by LY379268 was unaffected by BDNF cotreatment even in cells coexpressing TrkB and mGlu 2 (Fig. 5E) or mGlu 3 (Fig. 5F).Collectively, these results suggest that TrkB stimulation does not affect G protein recruitment to mGlu 2 but stabilizes mGlu 2 in an active conformation that promotes G protein activation in the absence or presence of agonist.
To further confirm these findings in an authentic tissue context, we performed a time-resolved Förster resonance energy transfer (trFRET)-based guanosine 5′-O-(3′-thiotriphosphate) (GTP-γ-S) binding assay in PFC membrane preparations.This assay relies on trFRET-based conformational sensors that recognize the active GTPγ-S-bound state of endogenous Gα i proteins and any conformation of Gα i proteins, respectively (Fig. 5G).Upon agonist stimulation of GPCRs, the guanine nucleotide exchange activity of the receptor promotes the release of GTP-γ-S from activated Gα i proteins, leading to a decrease in trFRET.Incubation of membranes with LY379268 induced a concentration-dependent decrease in the trFRET signal, indicative of nucleotide release from Gα i proteins induced by the agonist-bound mGlu 2 receptor (Fig. 5H).Pretreatment of membranes with 1 μM 7,8 DHF enhanced both basal and LY379268-induced response (Fig. 5H).These findings are reminiscent of our observations in HEK-293 cells showing that TrkB activation enhances mGlu 2mediated G protein activation.Together with data indicating that mGlu 2 activation transactivates TrkB in mouse cortical neurons and PFC, they demonstrate a reciprocal cross-talk between native mGlu 2 and TrkB.

TrkB pharmacological inhibition prevents behavioral deficit alleviation by glutamatergic antipsychotics in mice treated subchronically with PCP
Mice subchronically treated with PCP (10 mg/kg, s.c.injection for five consecutive days during 2 weeks) (24) were used to explore the role of TrkB in the behavioral response to mGlu 2 stimulation.Consistent with previous studies (24), PCP-treated mice showed deficits in short-term (Fig. 6A) and long-term (Fig. 6B) memory in the novel object recognition task.Acute administration of LY379268 (10 mg/kg, 30 min, i.p.) or 7,8-DHF (5 mg/kg, 1 hour, i.p.) before the test restored normal short-and long-term novelty discrimination in PCP-treated mice to levels similar to those observed in vehicle-treated mice.LY341495 (3 mg/kg, 30 min, i.p.) or ANA12 (2.5 mg/kg, 2 hours, i.p.) did not affect the memory capacities of PCP-treated mice (Fig. 6, A and B) when they were injected alone but prevented the alleviation of memory deficits elicited by LY379268 and 7,8-DHF (Fig. 6, A and B), respectively, in PCP-treated mice.The ability of LY379268 to alleviate short-term and long-term memory deficits in PCP-treated mice was also prevented by the administration of ANA-12, indicating a key influence of TrkB activation in the procognitive effect of the mGlu 2 agonist.Neither LY341495 nor ANA-12 nor LY379268 (alone or in combination with LY341495 or ANA-12) nor 7,8-DHF (alone or in combination with ANA-12) altered shortterm (fig.S8A) or long-term (fig.S8B) memory performance of vehicle-treated mice.None of the treatments significantly affected locomotor activity of mice (fig.S9).
We next examined the role of mGlu 2 /TrkB stimulation on immobility in the forced swim test (FST).This test is classically used as a model of depression and despair to screen for antidepressants, which decrease immobility time upon acute administration.Subchronic administration of NMDA receptor antagonists induce an increase in immobility time in the FST that is not affected by imipramine and likely does not reflect a depressive-like state (25,26).However, it is prevented by administration of atypical antipsychotics and is thus considered as a model of the negative symptoms of schizophrenia, such as reduced motivation and affective flattening (25).As expected, LY379268 or 7,8-DHF administration decreased the immobility time of PCP-treated mice to a level similar to that measured in vehicle-treated mice (Fig. 6C) (26).Injection of LY341495 or ANA-12, which alone had no effect on the immobility time of PCP-treated mice, prevented the rescue of PCP-induced motivation deficits by LY379268 and 7,8-DHF, respectively, in these mice.Reminiscent to its procognitive effects in the novel object recognition task, ANA-12 administration also abolished the rescue of PCP-induced motivation deficits (Fig. 6C), arguing for a key role of TrkB activation in the motivational effects of the mGlu 2 agonist in PCP-treated mice.Administration of LY341495 or ANA-12 or LY379268 (alone or in combination with LY341495 or ANA-12) or 7,8-DHF (alone or in combination with ANA-12) did not modify immobility time of vehicle-injected mice in the FST (fig.S8C).

DISCUSSION
While antipsychotics currently prescribed in patients with schizophrenia mainly target dopaminergic and serotonergic systems, a large body of evidence supports the notion that hyperfunction of the glutamatergic system has a key influence in the pathogenesis of the disease (27).This provided the impetus for the development of a recent generation of antipsychotics that behave as agonists or positive allosteric modulators of mGlu 2 .However, the neuronal signaling pathways and molecular targets that underlie their antipsychotic effects remain poorly characterized.One important issue is the high sequence homology between mGlu 2 and mGlu 3 (28), which makes highly challenging the design of mGlu 2 -specific ligands and, consequently, the characterization of signaling mechanisms specifically engaged by mGlu 2 , but not by mGlu 3 .
Taking advantage of our recently developed selective mGlu 2 nanobody ( 14), we provide a first unbiased characterization of mGlu 2 interactome in mice PFC.Analysis of protein-protein interactions between the 149 candidate mGlu 2 partners previously reported in public databases revealed that more than two-third of them are part of a unique protein complex.This indicates that the protocol used to purify mGlu 2 partners did not affect protein-protein interactions and outlines the relevance of our AP-MS strategy to get a global picture of the mGlu 2 interactome in vivo, including not only direct receptor partners but also proteins recruited via their association with intermediate partners.Further supporting the relevance of the results of our interactomic screen, it identified several proteins previously reported as mGlu 2 partners.These include the α subunit of the G i1 protein (GNAI1), consistent with the canonical coupling of mGlu 2 to G i/o proteins (11).Group II (mGlu 3 ) and group III (mGlu 4 , mGlu 7 , and mGlu 8 ) mGlu receptors, but not group I mGlu receptors (mGlu 1 and mGlu 5 ), were also identified in the mGlu 2 interactome.These findings suggest that mGlu 2 is colocalized with those receptors in some PFC neuronal populations, consistent with previous fluorescence in situ hybridization and single-cell RNA sequencing experiments indicating that most mGlu subtypes, with the exception of mGlu 6 , are coexpressed in the majority of cortical neurons.They also suggest that mGlu 2 can form heteromers with mGlu 3 , mGlu 4 , mGlu 7 , and mGlu 8 in PFC, corroborating previous observations using a trFRET assay in transfected cells indicating that group II and III mGlu receptors can heteromerize, whereas neither group II nor group III receptors form heteromers with group I receptors (16).Among possible heteromeric combinations, mGlu2-3 and mGlu2-4 heteromers have already been detected in mouse brain, especially the PFC (29,30).Furthermore, it has recently been proposed that mGlu 2 and mGlu 3 have a higher propensity to form heteromers compared to other mGlu receptor pairs, corroborating our results indicating that mGlu 3 is the most significantly enriched among mGlu receptors identified in the mGlu 2 interactome (30).The existence of mGlu 2 /mGlu 7 and mGlu 2 /mGlu 8 heteromers remains to establish in PFC, as one cannot rule out indirect recruitment mGlu 7 and mGlu 8 via formation of oligomeric mGlu receptor assemblies or association with different intermediate partners.
The specific identification of group II and III mGlu receptors in the mGlu 2 interactome also reflects their predominant localization in neurons: group II receptors, including mGlu 2 , and group III receptors are mostly found in presynaptic compartment, while group I mGlu receptors are almost exclusively post-synaptic (19).Nevertheless, there is compelling evidence indicating that a fraction of mGlu 2 is also located post-synaptically, where it can associate with the serotonin 5-HT 2A receptor, another major target of antipsychotics (31).It has been proposed that post-synaptic mGlu 2 /5-HT 2A heteromers constitutes an important target of antipsychotics (32) and that normal nonpsychotic state and the ability of 5-HT 2A and mGlu 2 ligands to alleviate psychotic symptoms depend on a homeostatic balance of G i/o -and G q -dependent signaling under the control of post-synaptic mGlu 2 /5-HT 2A heteromers (33,34).Analysis of functional annotations within the mGlu 2 interactome revealed a predominant presynaptic localization of the mGlu 2 -associated complex and a strong enrichment in biological processes and molecular functions characteristic of presynaptic receptors.Consistent with the absence of 5-HT 2A in our interactome, this suggests that our interactomic screen mostly identified the interactome of presynaptic mGlu 2 , while the interactome of post-synaptic receptor could not be identified, presumably due its lower abundance.
Our interactomic screen also identified subunits 2A and 2B of glutamate ionotropic receptor of N-methyl-d-aspartate types (GRIN2A and GRIN2B) that are expressed not only at the post-synapse as initially thought but also in the presynaptic compartment where they play a key role in shaping synaptic transmission and plasticity (35).A study has shown that activation of group II mGlu receptors promotes long-term potentiation induction at Schaffer collateral-CA1 pyramidal cell synapses through a mechanism involving enhancement of NMDA receptor-mediated currents, suggesting a functional cross-talk between mGlu 2/3 and NMDA receptors (36).The importance of physical interaction of NMDA receptor subunits with mGlu 2 and/or mGlu 3 in modulation of synaptic plasticity remains to be established.Likewise, given the key influence of NMDA receptor hypofunction in the pathophysiology of schizophrenia (37), the association GRIN2A and GRIN2B with presynaptic mGlu 2 and mGlu 3 in the context of schizophrenia certainly warrants further exploration.
Among the newly identified mGlu 2 -interacting proteins, we focused on the RTK TrkB that displayed a robust and reproducible enrichment in the mGlu 2 interactome, in view of (i) the decreased expression of TrkB and its natural ligand BDNF in patients with schizophrenia in comparison to general population ( 15), (ii) the association of BDNF Val 66 Met polymorphism that alters BDNF secretion and schizophrenia (38), and (iii) the ability of a specific TrkB agonist to reverse cognitive and synaptic plasticity deficits in a rat model of schizophrenia (39).Using PLA, we confirmed the association of TrkB with endogenous mGlu 2 in various brain regions including PFC, OB, DG, and CB, whereas TrkB did not interact with the other group II member mGlu 3 .This result was confirmed by BRET experiments, which also suggest that TrkB directly binds to mGlu 2 .Furthermore, TrkB-mGlu 2 interaction was promoted by agonist stimulation of either mGlu 2 or TrkB and inhibited by a mGlu 2 antagonist, indicating that it dynamically regulated by the conformational state of both receptors.This agonist-dependent physical complex formations between GPCRs and RTKs had only been demonstrated for a few receptor pairs (22), including AT1 angiotensin/epidermal growth factor receptor (40) and β 2 -adrenergic/ vascular endothelial growth factor receptor 2 (41) and never for a class C GPCR.
These observations prompted further studies exploring the reciprocal influence of both receptors on their signal transduction, which revealed that mGlu 2 stimulation induces TrkB activation, as assessed by its phosphorylation on Tyr 816 , in PFC neurons.Notably, TrkB phosphorylation level elicited by mGlu 2 activation was comparable to that induced by direct TrkB stimulation by an agonist, suggesting that it is physiologically relevant.Reciprocally, TrkB stimulation promoted G o activation upon coexpression of mGlu 2 in the absence or presence of a mGlu 2 agonist, indicating that TrkB can transactivate unliganded mGlu 2 .Transactivation of RTKs by GPCRs has been demonstrated for numerous GPCR/RTK pairs (22).For instance, previous studies have shown that TrkB is transactivated by adenosine A 2A receptor (42) and pituitary adenylate cyclase activating polypeptide (PACAP) receptor (43), a process underlying neurotrophic effects of adenosine and PACAP, while stimulation of mGlu 2 transactivates insulin-like growth factor 1 receptor (44).Although less extensively observed, RTK ligands can also transactivate GPCRs, indicating that transactivation can be bidirectional (22,45).To our knowledge, the present study provides the first example of reciprocal transactivation between a GPCR and a RTK.Several GPCR/RTK transactivation mechanisms have been described depending on receptor pairings and cell types.They can be ligand dependent and result from ligand release or cleavage of a proligand by a protease, as well as ligand binding to their cognate receptor.Such a mechanism was not involved in TrkB transactivation induced by LY379268, as it was not prevented by a BDNF neutralizing antibody that inhibited TrkB activation induced by exogenous BDNF.Bidirectional GPCR/ RTK transactivation can also result from the production of second messengers and activation of protein kinases upon activation of one of the receptors, which, in turn, promote phosphorylation on Ser/ Thr or Tyr residues and activation of the unliganded partner receptor.Last, it can involve formation of RTK/GPCR heterocomplexes, allowing complex allosteric regulations of both receptors (22,45).BRET experiments indicating a direct interaction between TrkB and mGlu 2 suggest that both receptors might behave as reciprocal positive allosteric modulators able to stabilize the partner receptor in an active conformation even in the absence of mGlu 2 agonist.
Notably, TrkB transactivation by mGlu 2 also requires activation of Gα i/o protein, since it was prevented by pretreatment of primary neurons with PTX.This suggests that additional partner proteins within the mGlu 2 /TrkB interactome might also contribute to the reciprocal transactivation of both receptors.
BDNF-TrkB signaling has long been considered a key player of neuronal development, synaptogenesis, as well as Hebbian and homeostatic plasticity (38).Over the two past decades, it has also emerged as critical factor underlying the effects of various psychiatric therapies (46).Both aminergic and fast-acting antidepressants promote the release of BDNF, and activation of TrkB signaling underlies their antidepressant effects despite the distinct time course and mechanism of action of both categories of antidepressants (47).Intriguingly, antidepressant-like effects of mGlu 2 antagonists in mouse models of depression seem to involve BDNF release (48,49), whereas we show in the present study that mGlu 2 agonists transactivate TrkB in healthy mice through a distinct mechanism that is independent of BDNF release.Last, mood stabilizers such as lithium and valproic acid also increase BDNF production, and BDNF-TrkB signaling has been involved in the anti-manic effects of lithium (46).However, the role of BDNF-Trk signaling in the response to antipsychotics is much less documented.While typical antipsychotics generally reduced BDNF expression in the brain, atypical antipsychotics such as clozapine often increase BDNF expression (50).Furthermore, the effects of antipsychotics on BDNF/TrkB expression seem to depend on the brain region examined and the duration of the treatment (38,51).Although the influence of glutamatergic antipsychotics on BDNF release remains to be elucidated, we show in the present study a key role of TrkB activation in the ability of glutamatergic antipsychotics to alleviate cognitive and negative symptoms in mice treated subchronically with PCP, providing a first link between BDNF-TrkB signaling and therapeutic response to antipsychotics.As TrkB pharmacological inhibition affects both TrkB alone signaling and TrkB/mGlu 2 heteromer signaling, a tool that specifically disrupts TrkB/mGlu 2 interaction without affecting TrkB response to BDNF, would certainly provide valuable information about the relative contribution of direct TrkB/mGlu 2 transactivation versus more indirect signaling cross-talk mechanisms in the behavioral response to glutamatergic antipsychotics.The ability of BDNF-TrkB signaling to sustain or enhance neuroplasticity altered in schizophrenia might explain the improved therapeutic efficacy of glutamatergic antipsychotics compared to currently available monoaminergic antipsychotics in alleviating negative and cognitive symptoms of schizophrenia.
Primary cultures of cortical neurons were prepared from 16.5-day-old embryonic mice, as previously described (53).All treatments were performed after 14 days in culture [days in vitro (DIV14)].
The significance of each protein enrichment was calculated with a Student's t test (one-sided test, correction s0 = 0).
The list of proteins exhibiting a significant enrichment in mGlu 2 immunoprecipitates, compared with control immunoprecipitates (P value < 0.05), was used as input in String software (https://stringdb.org)for representation of protein-protein physical interactions previously reported with score of ≥0.4 in String databases.The list of proteins of the most connected network was uploaded in g:Profiler software (https://biit.cs.ut.ee/gprofiler/gost) to calculate enrichment in GO functional annotations against the whole list of proteins identifed in our experiments.
Immunoprecipitation from transfected HEK-293 cells and primary neurons HEK-293 cells were transfected with plasmids encoding human Flag-mGlu 2 or human Flag-mGlu 3 in combination or not of a plasmid encoding human TrkB or empty vector at 40 to 50% confluence using Lipofectamine 2000 (Invitrogen).Forty-eight hours after transfection, cells were starved of serum for 4 hours and lysed in the same ice-cold lysis buffer as for the AP protocol.The immunoprecipitation was performed with as previously described (54).Briefly, solubilized proteins (1.5 mg) were incubated with anti-Flag M2 affinity gel beads (Sigma-Aldrich, #A2220) for 4 hours at 4°C.Samples were centrifuged (5 min, 1000g) and washed three times with an ice-cold solution of 500 mM NaCl and phosphatase inhibitors and once with 150 mM NaCl and phosphatase inhibitors.Immunoprecipitated proteins were then eluted in Laemmli sample buffer.
Immunoprecipitation from primary cultured cortical neurons was performed using 0.5 mg of solubilized neuronal proteins and the mGlu 2 nanobody (2 μg) as described for the AP-MS procedure.Control immunoprecipitations were performed with a nonimmune nanobody.Samples were centrifuged (5 min, 1000g) and washed three times with an ice-cold solution of 500 mM NaCl and phosphatase inhibitors and once with 150 mM NaCl and phosphatase inhibitors.Immunoprecipitated proteins were then eluted in Laemmli sample buffer.

Bioluminescence resonance energy transfer
For saturation assay, cells were cotransfected with a constant amount of constructs encoding TrkB-Rluc or mGlu 5 -Rluc (20 ng per 100,000 cells, BRET donors) and increasing amounts of constructs encoding mGlu 2 -Venus or mGlu 3 -Venus or mGlu 2 C3-Venus or membrane bound CAAX-Venus or cytoplasmic Venus (from 0 to 40 ng per 100,000 cells, BRET acceptors).For G protein dissociation assay, cells were cotransfected with constructs encoding Gα o -Rluc (10 ng per 100,000 cells), Flag-Gβ (20 ng per 100,000 cells), and Gγ-Venus (20 ng per 100,000 cells) and constructs encoding human Flag-mGlu 2 (20 ng per 100,000 cells) or Flag-mGlu 3 (20 ng per 100,000 cells) in combination or not with a plasmid encoding human TrkB (20 ng per 100,000 cells), as indicated.For miniG protein recruitment assay, cells were cotransfected with construct encoding miniG si -Rluc chimeric protein (10 ng per 100,000 cells) and constructs encoding mGlu 2 -Venus or mGlu 3 -Venus (20 ng per 100,000 cells) in combination with a plasmid encoding human TrkB (20 ng per 100,000 cells), as indicated.In all experiments, cells were cotransfected with a plasmid encoding the plasma membrane glutamate transporter EAAC1 (6 ng per 100,000 cells) to maintain extracellular glutamate concentration at low levels and minimize mGlu receptor activation by endogenously released glutamate.Forty-eight hours after transfection, cells were starved of serum for 1 hour with DMEM + GlutaMAX and incubated for 10 min with coelanterazine H (5 μM final concentration) in Dulbecco's phosphate-buffered saline (PBS) (Thermo Fisher Scientific, reference no.14040083).For G protein dissociation and miniG recruitment assays, cells were stimulated for 10 min with increasing concentrations of LY379268 (10 −12 to 10 −5 M) in the presence or absence of 10 nM BDNF.BRET signals were measured using a Mithras LB940 instrument.Rluc8 emission was measured at 410 ± 80 nm, and Venus emission was measured at 515 ± 30 nm.Net BRET ratios were calculated as previously described (55) with the formula net BRET ratio = ratio [(Venus emission)/(Rluc8 emission)] in tested well − ratio [(Venus emission)/(Rluc8 emission)] in Rluc8 alone condition.

p-Y 816 -TrkB immunolabeling
For analysis of TrkB phosphorylation in primary cultures, cortical neurons cultured on glass coverslips were fixed in 4% paraformadehyde in PBS, permeabilized in PBS solution containing 5% BSA and 0.1% Triton X-100 for 30 min, and incubated with the anti-p-Y 816 -TrkB antibody (1:1000) overnight at 4°C in PBS containing 5% BSA and 0.1% Triton X-100.After three washes in PBS, cells were incubated in PBS containing 5% BSA and 0.1% Triton X-100 for 1 hour with an Alexa Fluor 488-conjugated anti-rabbit antibody (1:1000).After three washes in PBS, cells were incubated with DAPI (4 μM) for 5 min in PBS and washed again three times with PBS.Coverslips were mounted on superfrost ultra plus slides (Thermo Fisher Scientific, reference no.10417002) using fluorescent mounting medium (Dako, reference no.S3023).Immunofluorescence staining was imaged using an Axio Imager Z1 microscope equipped with apotome (Carl Zeiss).Image analysis was performed with Fiji software (v2.1.0),and results are expressed as the number of anti-p-Y 816 -TrkB-positive pixels per field (RawIntDen).
For analysis of TrkB phosphorylation in mouse PFC, mice were injected with 150 μl of Euthasol Vet (33 mg/kg; diluted in 0.9% NaCl, TVM Laboratory Innovative Animal Health).Five min after injection, mice were intracardially perfused with a fixing solution containing 4% paraformaldehyde, PBS (pH 7.5), 10 mM sodium fluoride, and 2 mM sodium orthovanadate.Brains were post-fixed for 48 hours in the same solution and successively transferred to PBS containing 10, 20, and, lastly, 30% sucrose.Coronal slices (30-μm thick) were cut with a Vibratome (Leica).Epitopes were first unmasked for 30 min with 1% sodium borohydride in PBS.After three washes, slices were permeabilized in PBS solution containing 5% goat serum, 5% horse serum, and 0.1% Triton X-100 for 2 hours, as previously described (56).Slices were then incubated with the anti-p-Y 816 -TrkB antibody (1:500) for 48 hours at 4°C.After three washes in PBS, they were incubated with an Alexa Fluor 488-conjugated anti-rabbit antibody (1:500) for 90 min in PBS solution containing 5% goat serum, 5% horse serum, and 0.1% Triton X-100.After three washes, slices were incubated 5 min in PBS with DAPI (4 μM) and washed again three times.Slices were mounted on superfrost ultra plus slides (Thermo Fisher Scientific, reference no.10417002) using fluorescent mounting medium (Dako, reference no.S3023).Immunofluorescence staining was imaged using a TCS SP8 X confocal microscope (Leica).Image analysis was performed using Fiji software (v2.1.0),and results are expressed as the number of anti-p-Y 816 -TrkBpositive pixels per field (RawIntDen).

Immunolabeling with anti-mGlu 2 nanobody
Prefrontal cortex coronal sections were incubated for 1 hour at 20°C with a blocking solution (3% BSA and 0.1% Triton X-100 in PBS).Sections were then incubated with d2-labeled anti-mGlu 2 nanobody (200 nM in the blocking solution) overnight at 4°C and were washed with PBS and distilled water.Fluoroshield with 4′,6-diamidino -2-phenylindole (DAPI) (Sigma-Aldrich) was used as mounting medium to preserve the fluorescence of brain sections.Images were obtained with a slide scanner Axio scan Z1 microscope (Carl Zeiss Microscopy) by performing full-section mosaics at ×20 magnification.

Proximity ligation assay
For experiments performed in cell cultures, HEK-293 cells were incubated with anti-TrkB (1:1000) and anti-Flag (1:1000) antibodies overnight at 4°C.For experiments in mice brain, sagittal brains slices (16-μm thick) were cut with a cryostat (Leica).Slices were incubated with anti-TrkB (1:500), anti-6-His (1:500) antibodies, and anti-mGlu 2 nanobody (200 nM) for 48 hours at 4°C.PLA labeling was then performed according to the manufacturer's instructions (Duolink In Situ Detection Reagents Orange, Sigma-Aldrich).HEK-293 cells and brain slices were imaged on a confocal microscope (Leica TCS SP8 X).All acquisitions were made the same day under the same confocal laser power settings to minimize variability among samples.Quantitative analysis of PLA complexes was performed as previously described (57).

trFRET assay for native G i protein activation
Prefrontal cortices of 2-month-old mice were homogenized with a Dounce homogenizer in 1 ml of homogenization buffer containing 25 mM tris HCl (pH 7.4), 10 mM MgCl 2 , 0.5 mM EDTA, 10 mM MgCl 2 , 10% sucrose, and cOmplete protease inhibitor cocktail (Roche).Tissue debris were eliminated by a 10-min centrifugation at 1500g.Supernatants were centrifuged again for 15 min at 13,300g to pellet membranes.Membranes were then resuspended in 500 μl of homogenization buffer, snap-frozen, and stored at −80°C.The assay was performed in 386-well plates (ProxiPlate 384 Plus, PerkinElmer).All reagents used for the trFRET assay were diluted in trFRET Taglite buffer (Cisbio Bioassays Revvity).Before the assay, the biotinylated PKB53 peptide (50 nM; Cisbio Bioassays Revvity), which specifically binds to the active conformation of Gα i proteins, which releases bound GTP-γ-S upon GPCR activation, was pre-incubated with 25 nM streptavidin-XL665 (trFRET acceptor, Cisbio Bioassays Revvity) for 30 min at 20°C.For the assay, 2.5 μg of PFC membranes was incubated for 10 min at 20°C with increasing concentrations of LY379268 in the presence or absence of 1 μM 7,8-DHF in the trFRET Tag-lite buffer supplemented with 1 nM GTP-γ-S.The XL665-PKB53 complex and a terbium-labeled Ab13 antibody (trFRET donor, 0.25 nM; Cisbio Bioassays Revvity) recognizing all conformations of Gα i1,3 proteins were then added for 3 hours at 20°C.The trFRET signal was measured using a PHERAstar microplate reader (BMG LABTECH) with the following setup: excitation at 337 nm (40 flashes per well), donor emission at 620 nm, acceptor emission at 665 nm, integration delay of 60 μs, and integration time of 400 μs.

Behavioral studies Object recognition test
This task was performed as previously described (58).On day 1, mice were habituated to the arena for 10 min.On day 2, they were subjected to a 5-min training session with two similar objects (dices or Lego plastic toys of approximately 3 cm in width and length and 5 cm in height), a 1-hour retention interval, and a 5-min test session in which one of the objects was replaced by a different object (shortterm memory).On day 3, mice were subjected to another 5-min test session in which the previously replaced object was replaced again by a novel object (long-term memory).The experiments were video-recorded, and exploration times (nose in contact or sniffing at less than 1 cm) were measured double blinded.Discrimination indexes were calculated using the formula [(exploration time of novel object − exploration time of familiar object)/total object exploration time].Forced swim test FST was performed as previously described (25).Testing was carried out in a 2-liter glass beaker (10 cm in diameter, 30 cm in height) filled with 1.4 liter of tempered water.Mice were left to swim for a 5-min test session.The experiments were video-recorded, and the immobility time was measured double blinded.Percentage of immobility during the 5-min session test was compared between groups.

Statistical analysis
Statistical analysis was performed using Prism (v.8.0, GraphPad Software Inc).Histograms or curves show means ± SEMs.Normal distribution of values was evaluated by Shapiro-Wilk normality test.Biochemistry and immunocytochemistry experiments were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett's test.PLA experiments were analyzed by two-way ANOVA followed by either Dunnett's test or Šidák's multiple comparison test.Immunohistochemistry and BRET experiments were analyzed by two-way ANOVA followed by Šidák's multiple comparison test.Behavioral experiments were analyzed by Kruskal-Wallis followed by Dunn's multiple comparisons test.For each comparison, F value, number of independent biological replicates, and sample size (or number of animals per condition) are indicated in the corresponding figure legends.

Supplementary Materials
This PDF file includes: Figs.S1 to S9 legends for tables S1 and S2 Other Supplementary Material for this manuscript includes the following: tables S1 and S2

Fig. 2 .
Fig. 2. TrkB interacts with mGlu 2 but not with mGlu 3 .(A) Western blots of anti-Flag immunoprecipitates obtained from heK-293 cells expressing human Flag-tagged mGlu 2 or mGlu 3 in the absence or presence of human trkB.inputs (lysate) and anti-Flag immunoprecipitates (iP) were probed with anti-Flag antibody to detect mGlu 2 and mGlu 3 , anti-trkB antibody, and with anti-GAPdh antibody.the data are representative of four independent experiments performed on different cultures.(B) Representative images of PlA experiments performed with anti-Flag and anti-trkB antibodies in heK-293 cells expressing either human Flag-tagged mGlu 2 or mGlu 3 in the absence or presence of human trkB.PlA signal is shown in yellow, cell nuclei are stained with 4′,6-diamidino-2-phenylindole (dAPi) in blue, and green fluorescent protein transfection control is shown in magenta.Scale bars, 5 μm.data are representative of four independent experiments performed on different cultures.(C) Representative images of PlA experiments performed with the anti-mGlu 2 nanobody and the anti-trkB antibody on OB, PFc, dG, and cB slices from wild-type (Wt) or mGlu 2 KO (mGlu 2 −/− ) mice.PlA signal is shown in yellow; cell nuclei are stained with dAPi in blue.Scale bars, 50 μm.the histogram shows means ± SeM of total PlA punctae per region of interest from 10 to 13 mice per condition.two-way analysis of variance (AnOvA) (compared between wild type and mGlu 2 −/− , post hoc Šidák's test): ***P < 0.001.(D) BRet saturation curves obtained from heK-293 cells expressing a fixed amount of Rluc-trkB or Rluc-mGlu 5 and increasing amounts of venus-mGlu 2 or venus-mGlu 3 or venus-mGlu 2 c3 (mGlu 2 with mGlu 3 c-terminal) or membrane bound cAAX-venus or cytoplasmic venus.curves show means ± SeM of net BRet signals measured in three technical replicates of one experiment representative of four biological replicates.two-way AnOvA (post hoc Šidák's test): F 8,26 = 638.6,***P < 0.001.

Fig. 6 .
Fig. 6.TrkB transactivation mediates rescue by a mGlu 2 agonist of behavioral deficits in PCP-treated mice.(A and B) Mice injected with either saline or PcP as described in Materials and Methods were pretreated with either vehicle or AnA-12 (2.5 mg/kg) or lY341495 (3 mg/kg; lY341) and then injected with either vehicle or lY379268 (10 mg/kg; lY379) or 7,8-dhF (5 mg/kg).the left histograms represent the means ± SeM of the percentage of exploration time of novel versus familiar object.the right histograms represent the means ± SeM of the discrimination index.each group included eight or more mice.Kruskal-Wallis (multiple comparisons compared to PcP vehicle, post hoc dunn's test): *P < 0.05 and ***P < 0.001.(C) Mice were treated as in (A) and (B). the histogram represents the percentage of immobility during the FSt.Kruskal-Wallis test (multiple comparisons compared to PcP vehicle, post hoc dunn's test): **P < 0.01 and ***P < 0.001.